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標題: | 含穿胞胜肽HBHAc之重組綠色螢光蛋白於不同酸鹼值下
之胞內運輸暨不同胜肽連接子對胞內運輸影響之研究 Study on the intracellular delivery of HBHAc-EGFP under different pH conditions and the effect of peptide linkers |
作者: | Pei-Yu Hsieh 謝佩妤 |
指導教授: | 沈麗娟 |
關鍵字: | 肝素結合凝血附著素,細胞穿膜胜?,胞內遞輸,綠色螢光蛋白,酸鹼值選擇性,胞吞作用,胜?連接子, heparin-binding haemagglutinin adhesion,cell-penetrating peptide,intracellular protein delivery,green fluorescence protein,pH sensitive,endocytosis,peptide linker, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 肝素結合凝血附著素 HBHA (heparin-binding haemagglutinin adhesion, HBHA) 為一源自於分枝桿菌表面的抗原蛋白,在本實驗室過去的研究中,我們發現由 HBHA 蛋白 C 端序列修改成之 HBHAc 是具有胞內遞輸能力的最小片段,接著,成功地將其應用於攜帶精胺酸去亞胺酶 (arginine deiminase, ADI) 、綠色螢光蛋白 (enhanced green fluorescent protein, EGFP) 以及細胞色素c (cytochrome c, cyt c) 至癌症細胞株內。此外,我們更發現 HBHAc-ADI 於人類乳癌細胞(MDA-MB-231)中表現出對酸鹼值敏感之遞送效果。因此,為了更進一步瞭解這個蛋白質載體對酸鹼值敏感的機轉以及找到 HBHAc 與其攜帶蛋白之間的最佳胜肽鍵結,本研究選擇綠色螢光蛋白作為模式蛋白,將 HBHAc 與綠色螢光蛋白利用融合蛋白或化學合成的方式連結,以綠色螢光蛋白的螢光活性評估 HBHAc 的遞送能力。而延續了本實驗室過去對於 HBHAc-EGFP 的研究,我們一樣選擇使用肝癌細胞(HepG2)作為體外試驗的給藥目標。
第一部份,我們利用大腸桿菌BL21 (DE3)菌株過度表現帶有 histidine-tag 之 EGFP 及 HBHAc-EGFP ,再使用 cOmplete His-Tag Purification Resin 作為親和性材料將蛋白純化。首先,我們將純化出的蛋白於不同酸鹼值的培養基環境下於 HepG2 細胞進行細胞攝取實驗,並使用流式細胞儀分析其進入細胞的效率。我們發現 HBHAc-EGFP 在不同酸鹼值下的細胞攝取量不同,當環境較為酸性時,其遞送進入細胞的效果會顯著增加,而EGFP在各種酸鹼值下被攝取入細胞的量都極少。MTT試驗結果則顯示此具有酸鹼選擇性之細胞遞送效果與不同酸鹼值之培養基之毒性沒有關聯性。 在證實 HBHAc-EGFP 的細胞遞送有酸鹼選擇性後,我們想進一步探討其進入細胞的機轉是否會受到於酸鹼值不同的影響,因此使用改變溫度或給予胞吞作用(endocytosis)化學抑制劑的方式來進行細胞攝取機轉方面的研究。我們發現在酸鹼值為 6.5 以及 7.5 下 HBHAc-EGFP 皆必須先與細胞膜表面的硫酸化醣蛋白結合後,再藉由胞吞作用進入細胞。然而,化學抑制劑的結果顯示,在酸鹼值為 6.5 時, HBHAc-EGFP 會透過由 dynamin 調節之胞吞作用被細胞攝取,而在酸鹼值為 7.5 ,其胞吞作用則受細胞膜表面之 lipid raft 影響。過去有研究指出物質之表面化學特性會影響其被攝取入細胞的機轉,因此我們推測其於不同酸鹼值下胞吞作用機轉的差異有可能就是因表面電荷的不同而造成。然而,雖然胞吞作用化學抑制劑的使用相當普遍,但其選擇性並不佳,因此,未來可能尚需要利用一些專一性較高抑制方式對於目前的結果做再次確認。 在第二部分的研究中,我們想要探討不同胜肽連接子對於 HBHAc 遞送效果的影響,使用了化學合成的方式將 HBHAc 或含有另外兩種不同胜肽連接子的 HBHAc 接在 EGFP 上。在酸鹼值為6.5時的結果顯示,與其他組別比起來,接上含有 flexible peptide linker 的 HBHAc 之 EGFP 被細胞攝取的量顯著的較低,因此flexible linker可能不適合用來連結 HBHAc 與 EGFP 。然而我們只有使用一種胜肽連接子的長度作測試,因此依照目前的研究結果,尚無法判斷rigid linker是否會對於細胞攝取的效率造成影響,故未來可能還要再嘗試不同長度的rigid linker以確認其效果。 HBHAc is the minimal segment of heparin-binding haemagglutinin adhesion (HBHA), the surface antigenic protein of Mycobacterium tuberculosis, with the ability of intracellular delivery. According to previous studies from our lab, HBHAc has been successfully utilized to deliver arginine deiminase (ADI), enhanced green fluorescent protein (EGFP), and cytochrome c (cyt c) into cancer cell lines. In addition, HBHAc-ADI has been demonstrated with a pH selective intracellular delivery to MDA-MB-231. Thus, to further investigate the mechanism of this pH selective carrier, and to optimize the peptide linkage between HBHAc and its functional protein cargo, EGFP was employed as the model protein and fused with HBHAc through either recombination or chemical conjugation. Based on the former study of HBHAc-EGFP from our lab, we selected the human hepatocellular carcinoma cell line, HepG2, as our in vitro model. For the first part of the study, recombinant histidine tagged EGFP and HBHAc-EGFP overexpressed in E. coli BL21 (DE3) were purified with cOmplete His-Tag Purification Resin. Uptake efficiencies of HBHAc-EGFP under different pH conditions were analyzed by flow cytometry. Results showed that when treated to HepG2 cells, HBHAc-EGFP showed a pH dependent cellular uptake, in which we observed that the internalization was higher under lower pH condition. The higher uptake efficiency of HBHAc-EGFP at pH 6.5 was not related to cytotoxicity, which was confirmed by MTT assay. Meanwhile, EGFP possessed very low permeability under all pH conditions. The mechanism of HBHAc-EGFP intracellular delivery under different pH conditions was investigated by the cellular uptake experiments under various conditions that each blocked a different component of an endocytosis pathway. Under both pH conditions, HBHAc-EGFP required binding to cell surface sulfated proteoglycans before internalized by endocytosis. However, the endocytic pathway of HBHAc-EGFP is different under pH 6.5 and 7.5. Results suggested that the internalization of HBHAc-EGFP in HepG2 cells was mediated by dynamin under pH 6.5, whereas it was related to lipid rafts under pH 7.5. It is possible that the different charges of HBHAc-EGFP under pH 6.5 and 7.5 resulted in the shift of its internalization mechanism. However, due to the lack of specificity of endocytosis inhibitors utilized, further confirmation with fluorescent tracers for specific endocytic pathways is required. For the second part of the study, HBHAc peptide with or without a peptide linker was chemically conjugated to EGFP to optimize the linkage type between HBHAc and EGFP. Reduced cellular uptake of EGFP conjugated with (GGGGS)2-HBHAc was observed under pH 6.5. This may be that the increased flexibility of fusion protein introduced by a flexible linker led to decreased intracellular delivery activity. Therefore, flexible linkers, such as (GGGGS)2, might not be suitable peptide linkers for HBHAc-EGFP. We are not yet able to confirm whether the addition of rigid linkers would affect the intracellular delivery efficiency of HBHAc-EGFP, due to only one linker length was utilized in this study. Thus, further confirmation with increased peptide linker lengths is needed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59807 |
DOI: | 10.6342/NTU201700437 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥學系 |
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